Nervous system development depends on the proliferation and differentiation of neural stem cells that give rise to both neurons and glia. Stem cells also contribute to neurogenesis and gliogenesis in adult brain. Thus great interest has become focused on better understanding stem biology as well as on the therapeutic potential of modifying CNS stem cell behavior in vivo or transplanting isolated stem cells. Indeed transplantation has provided a powerful tool for defining the developmental potential of stem cells and holds great promise as a therapeutic tool in a variety of age-related disorders. However, limitations in the currently available methodology for isolating and following stem cells after transplantation have hindered development of this field. Here we propose to develop a marking system for nestin lineage CNS stem cells in the mouse embryo that will allow their ready isolation and identification in transplanted brain using as a stem cell marker an enhanced cyan fluorescent protein tagged with a nuclear localization signal (ECFPnl). This system will utilize a binary transgenic system in which in one transgene the nestin promoter will activate the expression of Cre recombinase. Cre recombinase will in turn activate a second transgene containing ECFPnl driven by a ubiquitously expressed chicken p-actin promoter that can only be activated by excision of a 'stop' cassette flanked by ZoxP sites. In an extension of this approach we will also use a recently described system that utilizes the Lac operator-repressor system in transgenic mice to develop an inducible system for marking nestin lineage CNS stem cells in adult brain with ECFPnl. Collectively if successful these studies will provide a simple and permanent marking system for identifying and following CNS stem cells in both fetal and adult mouse brain.